WO2007124383A2

WO2007124383A2 - 1,1'-binaphthyl-based inhibitors of nad+-dependent deacetylase activity and sir2-family members

Info

Publication number: WO2007124383A2
Application number: PCT/US2007/067006
Authority: WO
Inventors: Richard H. Goodman; Clark C. Fjeld; Michael D. Jackson
Original assignee: Oregon Health & Science University
Priority date: 2006-04-19
Filing date: 2007-04-19
Publication date: 2007-11-01
Also published as: WO2007124383A3

Abstract

Aspects of the present invention provide novel compositions and methods for inhibiting NAD+-dependent deacetylase activity (e.g., SIR2) and for affecting and/or treating SIR2-related biological conditions and disorders including but not limited to cancer, HIV/AIDS, silenced genes, metabolism, apoptosis, aging, and conditions such as Malaria and infectious disease (e.g., Trypanosoma brucei (African sleeping sickness); Leishmaniasis (e.g., Leishmania infantum, etc.); Mycobacterium tuberculosis; and Anthrax). The novel compositions and methods comprising use of novel small molecule inhibitors of NAD+-dependent deacetylase activity (e.g., SIR2 inhibitors) that comprise a characteristic a 1,1 -binaphthyl core structure. The novel compounds and compositions provide surprisingly effective inhibitors of NAD+-dependent deacetylase activity, and have substantial therapeutic utility. Further aspects provide methods of treatment, comprising administration of a therapeutically effective amount of one or more of the disclosed compounds, and further comprising administration of an inhibitor of topoisomerase 2 (e.g., etoposide), an inhibitor of type I and/or II histone deacetylase (HDAC) (e.g., suberoylanilide hydroxamic acid (SAHA)), or both.

Description

1,1'-BINAPTHYL-BASED INHIBITORS OF NAD+-DEPENDENT DEACETYLASE ACTIVITY AND SIR2-FAMILY MEMBERS

FIELD OF THE INVENTION

Aspects of the present invention relate generally to novel compositions and methods for inhibiting NAD+-dependent deacetylase activity (e.g., SIR2) and for affecting and/or treating

SIR2-related biological conditions and disorders including but not limited to cancer, HIV/ AIDS, silenced genes, metabolism, apoptosis, aging, and conditions such as Malaria and infectious disease (e.g., Trypanosoma brucei (African sleeping sickness), Leishmaniasis (e.g., Leishmania infantum, etc.), Mycobacterium tuberculosis, and Anthrax)., said novel compositions and methods comprising use of novel small molecule SIR2 inhibitors that comprise a 1,1'- binahpthalene core structure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent

Application Serial Nos. 60/793,430, filed on 19 April 2006 and entitiled "1,1'-BINAPTHYL- BASED INHIBITORS OF NAD+-DEPENDENT DEACETYLASE ACTIVITY AND SIR2-

FAMILY MEM," and 60/884,131, filed 09 January 2007 of same title, both of which are incorporated by reference herein in their entirety.

BACKGROUND

Human SIR2. The yeast protein Sir2 (Silent Information regulator 2, Sirtuin) is a NAD+- dependant deacetylase and is the defining member of the Type III class of histone/protein deacetylases. SIR2 enzymes consume NAD+ in a deacetylation reaction to afford free lysine, nicotinamide, and O-Acetyl-ADPribose (both 2'- and 3'- regioisomers). Specifically, the acetyl group from the lysine e-amino group is transferred to the ADPribose moiety of NAD+, producing nicotinamide and the novel metabolite O-Acetyl-ADPribose, the function of which has yet to be elucidated. The Sirtuin protein class is conserved from bacteria to humans, of which 7 members have been identified but only three (SIRTl, SIRT2, and SIRT3) have demonstrated enzymatic activity in vitro or in vivo. The SIR2 family of deacetylases is involved in a variety of biological control systems including the control of gene silencing, metabolism, apoptosis, and aging. For example, the deacetylase activity of SIRTl, a human homologue of the yeast Sir2 enzyme, promotes tumorigenesis by opposing the proapoptotic activities of various protein substrates, including p53, Foxo3a, and BCL6.

p53, Foxo3a and BCL6. The tumor suppressor p53 is a critical regulator of cell death pathways. In response to a variety of genotoxic insults (e.g., DNA damage), p53 becomes activated and induces the expression of pro-apoptotic genes. This activity is required to eliminate cells that would otherwise go on to produce tumors. Additionally, the ability of chemotherapeutic agents to kill cancer cells in some instances depends on an intact p53 pathway. The activity of p53 depends, at least in part, on acetylation by CBP/p300, proteins originally identified in one of the applicants' labs, and such acetylation of p53 is reversed (inactivated via deacetylation on lysine 382) by SIRTl. In addition, SIRTl deacetylates and represses the activity of the forkhead transcription factor Foxo3a and other mammalian forkhead factors, and by restricting mammalian forkhead proteins, SIRTl also reduces forkhead- dependent apoptosis (Motta et al., Cell, 116:551-563, 2004). Additionally, the function of BCL6, a transcriptional repressor implicated in the pathogenesis of B-cell lymphomas, critically depends on its deacetylation by HDAC and SIR2-like enzymes (Bereshchenko et al., Nature Genetics 32:606-613, 2002). Therefore, the emerging role of SIRTl as an anti-apoptotic factor reflects its importance as an anticancer target (Figure 1).

Non-Hodgkin 's lymphomas. One particular class of tumor, non-Hodgkin's lymphomas (NHL), has an incidence in the U.S. of over 50,000 cases per year (about 5-fold greater than that of Chronic Myelogenous Leukemia (CML), which is also known as chronic myeloid or chronic myelocytic leukemia). Follicular lymphomas account for about 35% of NHL and, of these, 55% involve BCL6 (transcriptional repressor) over-expression. Diffuse large cell lymphomas account for 30-40% of NHL, and, of these, 30% have BCL6 abnormalities. Thus, BCL6 over- expression is responsible for about half (50%) of the cases of NHL, accounting for 25,000 new cases per year. NHL is frequently due to chromosomal translocations that induce expression (over-expression) of BCL6, which targets the p53 promoter and blocks p53 transcription, rendering the affected cell incapable of, inter alia, mediating an apoptotic response. This block of p53 expression is central to the oncogenic process in NHL. Significantly, BCL6 is also regulated by acetylation except, in contrast to the effect of acetylation on p53, acetylation blocks BCL6 action. Therefore, BCL6 is rendered inactive when it is maintained in an acetylated state, and when this occurs, p53 is derepressed and p53 levels rise. Remarkably, the acetylation state of BCL6 is also regulated by SIRTl; that is, inhibition of SIRTl promotes BCL6 acetylation and blocks its repressor function.

The current therapy for NHL is ChOP (cyclophosphamide, doxorubicin, vincristine, predisone) and Rituximab (chimeric anti-CD 20 monoclonal antibody). However, such therapy is not only expensive, but the treatment is moderately toxic and recurrence rates are high.

Moreover, such therapy does not engage the underlying metabolic pathways involving BCL6 and p53 described above.

HIV/AIDS. The TAT protein factor is a critical mediator for human immunodeficiency virus (HIV) transcription. Deacetylation of TAT is required for recycling, and maintenance of TAT in its acetylated state blocks such recycling. Significantly, SIRTl deacetylates the TAT protein factor, and is thus implicated in the progression of HIV infection and AIDS.

Inhibitors. Because of their role in a variety of cellular pathways (e.g., including those relating to cancer, HIV, gene silencing, metabolism, apoptosis, and aging), the identification of inhibitors to these histone/protein deacetylases has been an active area of research, and such inhibitors have been shown to induce growth arrest, differentiation, and/or apoptotic cell death in transformed cells. Some small molecule inhibitors of these deacetylases are known in the art. For example, splitomicin has been identified as a micromolar inhibitor of yeast Sir2, and analogues thereof have been identified as small molecule inhibitors through chemical screens (see, e.g., WO 03/046207 by Bedalov et al; and Posakony et al, J. Med. Chem. 47:2635-2644, 2004). In the case of splitomicin and analogues thereof, the hydrolytically unstable lactone ring of splitomicin is regarded as being critically important for activity (Posakony et al, supra).

Other known inhibitors are characterized as having a pharmacophore comprising a critical phenol moiety, together with a hydrophobic moiety and hydrogen-bonding features (Tervo et al., J. Med. Chem. 47:6292-6298, 2004). [Stuart Schreiber from the Chemical Biology Department at Harvard has taken a similar approach. In contrast, investigators at Albert Einstein have taken a different tack, utilizing peptides that interfere with BCL6 action.1 Significantly, however, the therapeutic efficacy of known inhibitors is limited, not only because of cellular permeability issues in particular instances, but also because such agents are active only when present at micromolar concentrations.

Therefore, there is a pronounced need in the art for identification of novel and more potent small molecule SIR2 inhibitors to provide novel compositions and methods for treatment of cancer including, but not limited to non-Hodgkin's lymphomas, and other SIR2-mediated disorders and conditions {e.g., HIV infection/ AIDS, gene silencing, metabolism, apoptosis, and aging). Particularly, there is a pronounced need in the art for small molecule SIR2 inhibitors that are efficacious at nanomolar concentrations. There is a pronounced need for SIR2 inhibitors having utility as an adjunct for other therapeutic modalities, such as utility for increasing the efficacy of targeted anticancer modalities {e.g., including, but not limited to chemotherapy and/or radiotherapy). There is a pronounced need in the art for SIR2 inhibitors having utility for activating expression of genes that are transcriptionally silenced by epigenetic or other processes {e.g., for inducing the expression of fetal hemoglobin genes for treatment of sickle cell anemia, or for activating expression of muscle genes in muscular dystrophies). There is a pronounced need in the art for better compositions and methods to treat NHL, including, for example, better SIRTl inhibitors to directly affect two critical components of the NHL oncogenic process; namely, BCL6 and p53.

SUMMARY OF THE INVENTION

Particular embodiments of the present invention provide small molecule inhibitors of NAD+-dependent deacetylase activity {e.g., SIR2 family members, human SIRTl) that are substantially more effective than any inhibitors previously characterized. The inventive compounds comprise a l,l'-binaphthyl core structure that provides for surprisingly effective inhibitors of NAD+-dependent deacetylase activity. The inventive compounds and compositions thereof have substantial utility for the treatment of cellular proliferative disorders, and other conditions or activities mediated by cellular NAD+-dependent deacetylase activity (e.g., SIR2 activity), including, but not limited to cancer, HIV, silenced genes, metabolism, apoptosis, and aging.

Particular embodiments of the present invention provide compounds of formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; - CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted C_1-6alkyl, and the pharmaceutically acceptable salts thereof.

Preferably, the compound is of formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7-membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof. Preferably, the compound is of formula III:

wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹; wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; wherein X is >C0, >S0₂ or >PO₂R⁹, wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; and wherein Y and Z are O, except where X is CO wherein exactly one of Y or Z is methylene; and the pharmaceutically acceptable salts thereof.

Preferably, the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as for II.

Preferably, the compound is of formula V:

wherein R >3 , r R>4 , X, Y and Z are as defined above for Formula III.

In particular embodiments, and with reference to Formula II above, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, - C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl.

Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi- 2-naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2,-naphthol, 6,6'- Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate.

Additional aspects of the present invention provide pharmaceutical compositions, comprising a compound of Formula I, II, III, IV or V as described herein. Such compositions comprise pharmaceutically acceptable carriers, diluents, and/or excipients, which are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Yet further embodiments provide a method for treating a cell proliferative disorder, comprising administering, in a subject in need thereof, a therapeutically effective amount of a compound of Formula I, II, III, IV or V as described herein. In particular embodiments, and with reference to Formula II above, R¹ and R² are -OH, wherein R³ and R⁴ are independently

5 halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl. Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'- Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)- l,l'-bi-2-naphthol, 6,6'-Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate, and combinations

O thereof.

In additional embodiments, the inventive methods further comprise administration of a chemotherapeutic or anti-neoplastic agent, as described herein above in relation to the inventive pharmaceutical compositions.

Preferably, the cell proliferative disorder is cancer, and preferably the cancer is selected

5 from the group consisting of Non-Hodgkins lymphoma, B-cell-derived Non-Hodgkins lymphoma, diffuse large B -cell lymphoma, CML, and combinations thereof.

Additional embodiments of the present invention provide methods for modulating a condition or activity mediated by cellular NAD+-dependent deacetylase activity, comprising contacting a cell having NAD+-dependent deacetylase activity with a NAD+-dependent

O deacetylase inhibiting amount of a compound of Formula I, II, III, IV or V as described herein.

In particular embodiments, inhibiting the NAD+-dependent deacetylase activity comprises inhibiting activity of a member of the SIR2 family of proteins with an inventive compound or composition as described herein above in relation to the compounds of Formulas I, II, III, IV and V, etc. Preferably, in such assays, modulating the condition or activity mediated

5 by cellular NAD+-dependent deacetylase activity comprises activating a silenced cellular gene. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises promoting p53-dependent apoptosis. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting BCL6 transcriptional repressor activity. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting HIV

TAT transcriptional activator activity.

Yet additional embodiments provide a method for treating or preventing HIV infection or HIV-related conditions (e.g., AIDS, Kaposi's sarcoma, etc.), comprising administering, in a 5 subject in need thereof, an effective amount of a compound of Formula I, II, III, IV or V as described herein. Preferably, with respect to Formula I, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl. Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'-Dibromo-l,l'-bi-2- O naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'-

Bis(trifluoromethyl)-l,l'-bi-2-naphthol, 6,6'-Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate, and combinations thereof. Preferably, the method further comprises administration of an anti-HIV agent selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non-nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (PIs); fusion 5 inhibitors (FI), and combinations thereof.

Yet further aspects provide methods of treatment (e.g.,:ce\\ proliferative disorders conditions (e.g., tumors); activities mediated by cellular NAD+-dependent deacetylase activity;

HIV infection or a HIV-related cellular effects, conditions or diseases, etc.), comprising administration of a therapeutically effective amount of a compound of the present invention, and O further comprising administration of an inhibitor of topoisomerase 2 (e.g., etoposide), an inhibitor of type I and/or II histone deacetylase (HDAC) (e.g., suberoylanilide hydroxamic acid

(SAHA)), or both.

Further aspects provide methods of treatment SIR2-related biological conditions and disorders including but not limited to cancer, HIV/ AIDS, silenced genes, metabolism, apoptosis, 5 aging, and conditions such as Malaria and infectious disease (e.g., Trypanosoma bruceϊ (African sleeping sickness), Leishmaniasis (e.g., Leishmania infantum, etc.), Mycobacterium tuberculosis, and Anthrax).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the involvement of SIRTl in the conversion of acetylated p53, Foxo3a and BCL6 to the respective deacetylated reaction products. In the absence of a SIR2 inhibitor (upper pathway) SIRTl -mediates conversion of acetylated p53, Foxo3a and BCL6 to the respective deacetylated reaction products, and cellular apoptosis (mediated through the p53, Foxo3a and BCL6 pathways) is terminated. Such conversion is precluded by a SIR2 inhibitor (lower pathway). According to particular aspects of the present invention, in the presence of an inventive SIRTl inhibitor, such cellular apoptosis is not terminated, because p53, Foxo3a and BCL6 remain acetylated.

Figure 2 illustrates how a SIR2 inhibitor should, in complementary fashion, affect two critical components of the non-Hodgkin's lymphoma (NHL) oncogenic process; namely, BCL6 and p53.

Figure 3 shows a comparison of the ability of several arginine derivatives to inhibit SIR2 at a concentration of 2 mM; from left to right, the bar graphs correspond to: reaction control; agmatine (decarboxylated arginine); L-Arginine; L-homo Arginine; L-Arg-Phe; and L-Arg-β- naphthyl. L-Arginine β-naphthylamide was found to be significantly better than the other arginine compounds tested, indicating, according to the present invention, the importance of the naphthyl ring for SIR2 binding.

Figure 4 illustrates, according to the present invention, similarities in the catalytic mechanism shared by SIR2 and PARP proteins. Figure 5 shows that at 200 μM, the PARP inhibitors APlOO and APlOl were significantly more effective at inhibiting SIR2 than the PARP inhibitors PJ34 and DPQ. According to the present invention the presence of a naphthyl moiety (e.g., as is present in the most effective PARP inhibitor APlOl) is important for SIR2 binding.

Figure 6 shows a comparison of SIR2-inhibition activity among a series of naphthyl containing compounds (at 286 μM) including disubstituted amino naphthols and binaphthol. According to the present invention, the compound containing two naphthol rings was significantly better at inhibiting SIR2 than the amino naphthol compounds.

Figure 7 shows a comparison of SIR2 inhibition activity among a series of binaphthyl compounds (at 143 μM) tested to further identify important determinants for SIR2 binding. SIR2 binding/inhibition activity was found to be sensitive to both the aromatic nature and the conformation of the binaphthyl ring system, and additionally was found to be sensitive to the identity of substituents at the 2, 2' positions.

Figure 8 shows that a binol derivative (dibromo naphthol; tested at 50 μM) containing strong electronegative bromine (Br) groups was found to be a particularly potent SIR2 inhibitor.

The influence of substituents on electron density of the naphthyl rings was found to be a major factor in SIR2 binding, and indicates that SIR2 prefers binaphthyl compounds that are more electron deficient.

Figures 9A and 9B show the results of H1299 cell-based assay for K382 acetylation of p53. Total p53 levels were measured using DOl antibody (p53 Total). The inventive compounds CBN and DBN increase p53 acetylation in intact H 1299 cells. Figure 9A shows that the IC50 values were < 1 μM.

Figure 10 shows an exemplary demonstration of the efficacy of the inventive compounds

{e.g., DBN) to inhibit BCL6 acetylation in Ramos cells, an art-recognized model of NHL. Figure 11 shows, according to particular aspects of the present invention, a dominant negative PfSIR2 generated by mutating catalytic Histidine to Tyrosine. GFP was replace by the mutant (PfSIR2 H 132Y) in an expression vector.

Figure 12 shows, according to particular aspects of the present invention, Northern blots probed with Exon 2 probe, which Hybridizes to conserved region present in many var genes (C=control; U=un-induced (+ATC); and I induced (-ATC). The Northern Blot revealed the induction of multiple var genes in both transfected cultures suggesting a lack of tetracycline control. Selective PCR using a primer specific to the mutant gene confirms the presence of the dominant negative PfSIR2 transcript in both the un-induced and induced cultures (compare "C" with "U" and "I"). Figure 13 shows, according to exemplary aspects of the present invention, selective PCR confirmation of expression of dominant negative PfSIR2 in transfected cultures. DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention provide novel compositions and methods for inhibiting NAD+-dependent deacetylase activity, and for affecting and/or treating protein deacetylase SIR2 family-mediated biological conditions and disorders, the methods including but not limited to those relating to cancer, gene silencing, metabolism, apoptosis, and aging, said novel compositions and methods comprising use of novel small molecule SIR2 inhibitors that comprise a characteristic l,l'-binaphthyl core structure that provides for surprisingly effective inhibitors of NAD+-dependent deacetylase activity.

Particular embodiments of the present invention provide small molecule inhibitors of SIR2 family members (e.g., human SIRTl) that are substantially more effective (e.g., about 50- fold or greater) than any inhibitors previously characterized (see, e.g., WO 03/046207 A2, describing small molecule lactone -based SIR2 inhibitors, and incorporated by reference herein in its entirety).

According to particular aspects, inhibition of SIR2 family activity (specifically SIRTl) promotes: p53 acetylation thereby increasing p53 activity, and promoting p53-dependent processes and conditions (e.g., apoptosis, activation of silenced genes, etc.); inhibition of BCL6 transcriptional repressor protein; enhancement of the efficacy of chemotherapeutic agents (e.g., antineoplastic agents); and provides methods for treating protein deacetylase SIR2 family- mediated biological conditions and disorders, the methods including but not limited to those relating to cancer, gene silencing, metabolism, apoptosis, and aging.

DEFINITIONS

The term "SIR2" refers to the art-recognized silent information regulator family of proteins, also known as sirtuins, and includes both mammalian and non-mammalian proteins (e.g., yeast homologs of SIR2 include HST1-4; mammaliam homologs include SIRT1-8, and sirtuins 1-8. Specific examples include, but are not limited to Sir2p and SIR2α (/zSIRTl)).

The term "apoptosis" refers to the art-recognized programmed/genetically determined cellular death, that is known to be affected by various cellular proteins. For example, the deacetylase activity of SIR2 promotes tumorigenesis by opposing the proapoptotic activities of various protein substrates, including p53, Foxo3a and BCL6.

The term "therapeutically effective amount" refers to that amount of the compound being administered sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the condition or disorder being treated.

The term "subject" refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

The phrase "HIV-related cellular effects, conditions and diseases" or "HIV-related (or mediated) conditions or diseases" refers to those illnesses and conditions included in, but not necessarily limited to the CDC 1993 AIDS surveillance case definition, as follows: Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer, invasive *; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5 C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HIV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1 month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HIV.

Particular compounds of the present invention comprise asymmetric carbon atoms {e.g., optical or chiral centers) or double bonds, and the racemates, diasteriomers, geometric isomers and individual isomers, enantiamers {e.g., (R) or (S)), etc., are all intended, according to particular aspects of the present invention, to be encompassed within the scope of the present invention. Additionally, in particular embodiments, isotopic variations, whether radioactive {e.g., ³H, ¹²⁵I, ¹³¹I, ¹⁴C, ³²P, ¹¹¹In, ⁹⁰Y, etc.) or not, are likewise intended to be encompassed within the scope of the present invention.

Identification of Novel Small Molecule Inhibitors of NAD+-dependent Deactylase Activity {e.g., SIR2 and human SIRTl family members)

As described herein under EXAMPLE 1, applicants have developed an HST2-based assay to identify novel SIR2 inhibitors. Two initial approaches described herein under EXAMPLE 2 were used by applicants to identify novel SIR2 inhibitors. First, transition state analogs were designed and tested based on applicants' perception and understanding of the catalytic mechanism of SIR2 family members {e.g., human SIRTl). Second, applicants identified novel inhibitors based on perception and exploitation of similarities in the catalytic mechanism of SIR2 and PARP proteins. This second approach was further facilitated by the availability of several PARP inhibitors.

Importance of the naphthyl ring moiety. Using these approaches, applicants herein disclose and demonstrate the importance of bi- and tri-cyclic ring systems, and in particular of the naphthyl ring moiety, in providing for effective small molecule inhibitors of SIR2. For example, FIGURE 4 and the respective description under EXAMPLE 2 herein below show applicants' demonstration of the importance of the naphthyl moiety for effective SIR2 inhibition {e.g., in the PARP inhibitor APlOl).

Surprising importance of the l,l '=binaphthyl moiety. Applicants, therefore, further investigated a series of naphthyl containing compounds, including disubstituted amino naphthols and binaphthol. Significantly, and surprisingly, compounds containing two naphthol rings in the form of a l,l'=binaphthyl core structure (e.g., (l,l'=binaphthyl)-2,2'-diol; compound 1) were significantly better at inhibiting HST2 ( a yeast Sir2 homologue) than the amino naphthol compounds (see, e.g., FIGURE 6, under EXAMPLE 3 herein below).

Surprising importance of both the aromatic nature and the conformation of the binaphthyl ring system, as well as the identity of the substituents at the 2, 2 ' positions of the l,l '=binaphthyl moiety. Applicants herein disclose and describe that substitutions of the binaphthalene moiety {e.g., 2, 2' substitutions) have a marked effect on the SIR2 inhibition activity (see, e.g., FIGURE 7 of EXAMPLE 4 herein below). Additionally, applicants herein demonstrate structure/activity relationships indicating that the influence of the substituents on the electron density of the naphthyl rings is a major factor in binding (FIGURE 7 of EXAMPLE 4). Significantly, binol derivatives containing strong electronegative groups (e.g., Br) were found to be among the most potent SIR2 inhibitor identified (see, e.g., FIGURE 8 of EXAMPLE 5 herein below).

Surprising determination of the importance of the influence of substituents on electron density of the naphthyl rings as a major factor in SIR2 binding. Applicants herein disclose that binol derivatives (e.g., dibromo naphthol) containing electronegative groups (e.g., mild electronegative groups) are particularly potent SIR2 inhibitors. For example, ring substitutions involving cyano groups, halogens and halogenated groups are herein disclosed as particularly useful in providing potent SIR2 inhibitors. The influence of binaphthyl ring substituents on electron density of the naphthyl rings was thus found to be a major factor in SIR2 binding, and indicates, according to particular aspects of the present invention that SIR2 prefers binaphthyl compounds that are more electron deficient (see, e.g., FIGURE 8 of EXAMPLE 5 herein below).

Broad Therapeutic Utility Inhibitors of S IR2 -mediated conditions or activities. According to the present invention (and see EXAMPLES 1-5 herein below), l,l'-binaphthyl-based compounds are preferred strong inhibitors of SIR2-mediated conditions or activities {e.g., inhibitors of SIRTl -mediated deacetylation of p53).

Inhibitors of S IR2 -mediated deacetylation of p53. Applicants herein show (EXAMPLE

6) that the inventive SIR 2 inhibitors {e.g., CBN: 6,6'-Dicyano-l,l'-bi-2,2'-naphthol, compound 5; or DBN: 6,6'-Dibromo-l,l'-bi-2,2'-naphthol, compound 2) substantially increase p53 acetylation in intact cells, thus supporting and confirming therapeutic utility of the inventive small molecules as effective inhibitors of NAD+-dependent deacetylase activity {e.g., hSIRTl and SIR2 family members (see, e.g., FIGURES 9A and 9B under EXAMPLE 6 herein below, showing that CBN and DBN increase p53 acetylation in intact H1299 cells)).

The inventive compounds, therefore, have substantial utility in novel compositions and methods for inhibiting NAD+-dependent deacetylase activity {e.g., SIR2) and for affecting and/or treating SIR2-related biological conditions and disorders including but not limited to cancer, HIV/AIDS (HIV-related cellular effects, conditions and diseases, silenced genes, metabolism, apoptosis, and aging.

Cancer; Non-Hodgkin's lymphomas. One particular class of tumor, non-Hodgkin's lymphomas (NHL) has an incidence in the U.S. of over 50,000 cases per year (about 5-fold greater than that of CML). NHL is the fifth most common cause of cancer in the United States, and the fifth leading cause of cancer death. Follicular lymphomas account for about 35% of NHL and, of these, 55% involve BCL6 (transcriptional repressor) over-expression. Diffuse large cell lymphomas account for 30-40% of NHL, and, of these, 30% have BCL6 abnormalities. Thus, BCL6 over-expression is responsible for about half (50%) of the cases of NHL, accounting for 25,000 new cases per year. NHL is frequently due to chromosomal translocations that induce expression (over-expression) of BCL6, which targets the p53 promoter and blocks p53 transcription, rendering the affected cell incapable of, inter alia, mediating an apoptotic response. This block of p53 expression is central to the oncogenic process in NHL. Significantly, BCL6 is also regulated by acetylation except, in contrast to the effect of acetylation on p53, acetylation blocks BCL6 action. Therefore, BCL6 is rendered inactive when it is maintained in an acetylated state, and when this occurs, p53 is derepressed and p53 levels rise. Remarkably, the acetylation state of BCL6 is also regulated by SIRTl; that is, inhibition of SIRT12 promotes BCL6 acetylation and blocks its repressor function.

The current therapy for NHL is ChOP (cyclophosphamide, doxorubicin, vincristine, predisone) and Rituximab (chimeric anti-CD 20 monoclonal antibody). However, such therapy is not only expensive, but the treatment is moderately toxic and recurrence rates are high. Moreover, such therapy does not engage the underlying metabolic pathways involving BCL6 and p53 described above.

By contrast, a SIR2 inhibitor (SIRTl inhibitor) should directly affect two critical components of the NHL oncogenic process; namely, BCL6 and p53 (see FIGURE X). According to preferred aspects of the present invention, this convergence of potential drug targets in a single oncogenic pathway provides an ideal scenario for development of therapeutic agents, because NHL should be particularly sensitive to the actions of SIR2 inhibitors, and in particular to the present novel SIR2 inhibitors that are substantially more active than those of the prior art.

Significantly, the results disclosed herein under EXAMPLE 7 below (see FIGURE 10), demonstrate the substantial utility of the inventive compounds in inhibiting BCL6 acetylation in Ramos cells, and art-recognized model of NHL.

HIV/AIDS. According to particular aspects, the inventive l,l'-binaphthyl-based compounds have substantial utility for treatment and/or prevention of HIV-related cellular effects, conditions and diseases, including treatment and/or prevention of AIDS.

The TAT protein factor of human immunodeficiency virus (HIV) is essential for the transcription activation (through TAR, an RNA stem-loop at the 5' end of viral transcripts) of the integrated HIV-I pro virus, and is a critical mediator for human immunodeficiency virus (HIV) transcription (within TAT transcription elongation is inefficient, resulting in abortive transcripts). Deacetylation of TAT is required for recycling, and maintenance of TAT in its acetylated state blocks such recycling. Significantly, Sir2 (SIRTl) deacetylates the TAT protein factor, and is thus implicated in the progression of HIV infection and AIDS. Therefore, according to additional aspects of the present invention, HIV infection/ AIDS and HIV-related cellular effects, conditions and diseases should be sensitive to the actions of SIR2 inhibitors, and in particular to the present novel SIR2 inhibitors that are substantially more active than those of the prior art (see, e.g, Pagans, Sara, et al., PIoS Biology 3:0001-0011, 2005, e41; incorporated by reference herein in its entirety).

The CD4+ T-lymphocyte coordinates a number of important immunologic functions, and a loss of these functions results in progressive impairment of the immune response. Studies of the natural history of HIV infection have documented a wide spectrum of disease manifestations, ranging from asymptomatic infection to life-threatening conditions characterized by severe immunodeficiency, serious opportunistic infections, and cancers. Other studies have shown a strong association between the development of life-threatening opportunistic illnesses and the absolute number (per microliter of blood) or percentage of CD4+ T- lymphocytes. As the number of CD4+ T-lymphocytes decreases, the risk and severity of opportunistic illnesses increase.

Therefore, according to aspects of the present invention, the inventive l,l'-binaphthyl- based compounds have substantial utility of treatment or prevention of HIV infection and/or replication and HIV-related cellular effects, conditions and diseases. The phrase "HIV-related cellular effects, conditions and diseases" or "HIV-related (or mediated) conditions or diseases" refers to those illnesses and conditions included in, but not necessarily limited to the CDC 1993

AIDS surveillance case definition, as follows: Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer, invasive *; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5 C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HIV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1 month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HIV.

Preferred Compounds

Particular aspects of the present invention provide a compound of formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; - CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

Preferably, the compound is of formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7-membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl, and the pharmaceutically acceptable salts thereof. Preferably, the compound is of formula III:

wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹; wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; wherein X is >C0, >S0₂ or >PO₂R⁹, wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl; and wherein Y and Z are O, except where X is CO wherein exactly one of Y or Z is methylene; and the pharmaceutically acceptable salts thereof.

Preferably, the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as in formula II.

Preferably, the compound is of formula V:

wherein R >3 , r R>4 , X, Y and Z are as defined above for Formula III.

In particular embodiments, and with reference to Formula II above, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, - C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl.

Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi- 2-naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'- Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate.

Preferred Compositions and Pharmaceutical Compositions

Additional aspects of the present invention provide a pharmaceutical composition, comprising a compound of Formula I, II, III, IV or V as described herein. Such composition comprises pharmaceutically acceptable carriers, diluents, and/or excipients; namely, that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Preferably, and with reference to Formula I above, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted C_1-6alkyl. Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'-Dibromo-l,l'-bi-2- naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'-Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate.

In alternate embodiments, the compositions additionally comprise an amount of an antineoplastic agent. Preferably, the antineoplastic agent is selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites, antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological response modifiers, DNA- alkylating agents, DNA intercalators, DNA repair agents, growth factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disrupters and topoisomerase I and II inhibitors, and combinations thereof.

Preferably, the antineoplastic agent is selected from the group consisting of cyclophosphamide, triethylenephosphoramide, triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine, trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU, benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil, cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin, estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide, improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine, vincristine, vinorelbine and vindesine.

Methods for Treating a Cellular Proliferative Disorder

Additional aspects of the present invention provide a method for treating a cell proliferative disorder, comprising administering, in a subject in need thereof, a therapeutically effective amount of a compound of Formula I:

Preferably the compound is of Formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7-membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl, and the pharmaceutically acceptable salts thereof. Preferably the compound is of Formula III:

wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; wherein X is >C0, >S0₂ or >PO₂R⁹, wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl; and wherein Y and Z are O, except where X is CO wherein exactly one of Y or Z is methylene; and the pharmaceutically acceptable salts thereof. Preferably, the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as described above for formula II. Preferably, the compound is of Formula V:

wherein R >3 , r R>4 , X, Y and Z are as defined above in relation to Formula III.

Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi- 2-naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'- Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate, and combinations thereof.

Preferably, the cell proliferative disorder is cancer, and preferably the cancer is selected from the group consisting of Non-Hodgkins lymphoma, B-cell-derived Non-Hodgkins lymphoma, diffuse large B -cell lymphoma, CML and combinations thereof.

In yet further embodiments the inventive methods further comprise administration of an chemotherapeutic or antineoplastic agent, as described herein above in relation to the inventive pharmaceutical compositions.

Methods for Modulating a Condition or Activity Mediated by Cellular NAD+-dependent Deacetylase Activity, and Exemplary Applications Thereof

Additional embodiments of the present invention provide methods for: modulating a condition or activity mediated by cellular NAD+-dependent deacetylase activity; activating a silenced cellular gene; promoting p53-dependent apoptosis; and inhibiting BCL6 transcriptional repressor activity.

Alternate embodiments provide a method for modulating a condition or activity mediated by cellular NAD+-dependent deacetylase activity, comprising contacting a cell having NAD+- dependent deacetylase activity with a NAD+-dependent deacetylase inhibiting amount of a compound of Formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; - CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl, and the pharmaceutically acceptable salts thereof.

Preferably, the compound is of formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7-membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl, and the pharmaceutically acceptable salts thereof; and whereby modulating of a condition or activity mediated by cellular NAD+-dependent deacetylase activity is afforded.

Preferably, the compound is of formula III:

wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; wherein X is >C0, >S0₂ or >PO₂R⁹, wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl; and wherein Y and Z are O, except where X is CO wherein exactly one of Y or Z is methylene; and the pharmaceutically acceptable salts thereof. Preferably, the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as in Formula II above. Preferably, the compound is of formula V:

wherein R >3 , r R>4 , X, Y and Z are as defined in relation to Formula III above.

In preferred embodiments, and in relation to Formula II above, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, - C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl.

In additional embodiments of the method for modulating a condition or activity mediated by cellular NAD+-dependent deacetylase activity, inhibiting the NAD+-dependent deacetylase activity comprises inhibiting activity of a member of the SIR2 family of proteins with an inventive compound or composition as described herein above in relation to the compounds of Formulas I, II, III, IV and V, etc.

Preferably, in such assays, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises activating a silenced cellular gene. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises promoting p53-dependent apoptosis. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting BCL6 transcriptional repressor activity. Alternatively, modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting TAT- mediated transcriptional activator activity in the context of HIV.

Yet additional embodiments provide a method for treating or preventing HIV infection or HIV-related conditions, comprising administering, in a subject in need thereof, an effective amount of a compound of Formula I, II, III, IV or V as described herein. Preferably, with respect to Formula II, R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl. Preferably, the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi- 2-naphthol, 6,6'-Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2- naphthol, 6,6'-Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate, and combinations thereof.

Preferably, the method further comprises administration of an anti-HIV agent selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non- nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (PIs); fusion inhibitors (FI), and combinations thereof.

Preferably, the reverse transcriptase inhibitor (NRTI) is selected from the group consisting of: lamivudine and zidovudine; FTC, emtricitabine; lamivudine, 3TC; abacavir/ lamivudine; zalcitabine, ddC, dideoxycytidine; zidovudine, AZT, azidothymidine, ZDV; abacavir, zidovudine, and lamivudine; tenofovir disoproxil/emtricitabine; enteric coated didanosine; didanosine, ddl, dideoxyinosine; Didanosine (ddl) delayed release capsules; tenofovir disoproxil fumarate; stavudine, d4T; abacavir, and combinations thereof.

Preferably, the non-nucleoside reverse transcriptase inhibitor (NNRTI) is selected from the group consisting of: delavirdine, DLV; efavirenz; nevirapine, BI-RG-587, and combinations thereof.

Preferably, the protease inhibitor (PI) is selected from the group consisting of: Amprenavir; indinavir, IDV, MK-639; saquinavir mesylate, SQV; saquinavir; lopinavir and ritonavir; Fosamprenavir Calcium; ritonavir, ABT-538; atazanavir sulfate; nelfinavir mesylate, NFV, and combinations thereof.

Preferably, the fusion inhibitor (FI) comprises enfuvirtide, T-20.

Administration, Formulation and Dosage of Inventive Compounds and Compositions

As will be appreciated by those of skill in the relevant art, an effective amount of the inventive compositions is determinable by the existence, nature, and extent of any adverse side- effects that accompany administration of the composition; the LD50 of the composition; and the side-effects of the composition at various concentrations. Typically, the amount of the composition administered will range from about 0.01 to about 20 mg per kg, more typically about 0.05 to about 15 mg per kg, even more typically about 0.1 to about 10 mg per kg body weight.

The compositions can be administered, for example, by intravenous infusion, orally, intraperitoneally, or subcutaneously. Oral administration is the preferred method of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

The compositions of the present invention are typically formulated with a pharmaceutically acceptable carrier, diluent or excipient before administration to an individual or subject. Pharmaceutically acceptable carriers, diluents or excipients are determined, in part, by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17^th ed., 1989; incorporated herein by reference).

Formulations suitable for oral administration can consist of: liquid solutions, such as an effective amount of the compound of Formula I, II, III, IV or V as described herein, suspended in diluents, such as water, saline or PEG 400; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; suspensions in an appropriate liquid; and suitable emulsions. Tablet forms can include one or more of the following agents: lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, for example, sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

The compositions of the present invention may be in formulations suitable for other routes of administration, such as, for example, intravenous infusion, intraperitoneally, or subcutaneously. The formulations include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response to the patient over time. For example, if the compositions of the present invention are administered to treat or prevent cancer, such as a tumor, the dose administered to the patient should be sufficient to prevent, retard, or reverse tumor growth. The dose will be determined by the efficacy of the particular composition employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular composition in a particular patient.

COMPOUNDS

The compounds of the present invention are synthesized by utilizing several methods known to one of ordinary skill in the art. Illustrative methods for preparing particular inventive species of the inventive compound genera are schematically shown below (Schemes 1, 2, 3 and

4 of the following pages), where various compounds are numbered for reference herein (see

TABLE 1 for list of corresponding exemplary compound names), but where the scheme is not intended to represent a comprehensive summary of all methods that can be used according to the present invention to synthesize the inventive compounds. For example, as appreciated by those of skill in the art, the starting materials {e.g., (l,l'-bi-2-naphthol (BINOL), compound 1; or 6,6'-

Dibromo-l,l'-bi-2-naphthol, compound 2), reagents and reactions shown in the following schemes can be appropriately modified to synthesize the various inventive compound species.

Particular compounds of the present invention comprise asymmetric carbon atoms {e.g., optical or chiral centers) or double bonds, and the racemates, diasteriomers, geometric isomers and individual isomers, enantiamers {e.g., (R) or (S)), etc., are all intended, according to particular aspects of the present invention, to be encompassed within the scope of the present invention. Additionally, in particular embodiments, isotopic variations, whether radioactive

{e.g., ³H, ¹²⁵I, ¹³¹I, ¹⁴C, ³²P, ¹¹¹In, ⁹⁰Y, etc.) or not, are likewise intended to be encompassed within the scope of the present invention.

Methods relating and/or applicable to the preparation of particular inventive species of the inventive compound genera are known in the art. For example Dong, C, et al.

(Tetrahedron: Asymmetry 11:2449-2454, 2000) discusses the synthesis of two different major groove chiral polymers of l,l'-binapthol, with polymerization occurring at the 6,6' positions, and use of such chiral polymers to effectively direct the enantio selective addition of diethylzinc to aldehydes to give high yields of chiral alcohols. Gao,Y., et al. (Journal of Medicinal Chemistry 44:2869-2878, 2001) discusses the identification of 5-carboxy-2-naphthoic acid (i.e., 6-carboxy-l -naphthoic acid) as a novel phenylphosphate mimetic and protein-tyrosine phosphatase (PTP; PTPlB) inhibitor by using a peptide mimetic platform, and discusses the synthesis of a variety of naphthyl compounds in this context. Bindal & Katzenellenbogen (Journal of Organic Chemistry 52:3181-3185, 1987) discuss exploitation of the ortho-metalating effect of the N,N-dialkylcarboxamide group in preparing c-1 alkyl- substituted 2,6-acylnaphthols by treating various naphthalenecarboxamides with n-BuLi or t-BuLi in tetrahydrofuran, followed by quenching with ethyl iodide to produce either ketones or C-1-ethylated naphthalenecarboxamides. Aoyama, T., et al (Chemical & Pharmaceutical Bulletin 33:1458- 1471, 1985) discuss the synthesis of various amidinonaphthols (by preparing nitriles, followed by conversion to imidates, and then to the amidinonaphthols) and of their acyl derivatives, as well as discussing evaluation of amidinonaphthols and acyl derivatives thereof as protease inhibitors (e.g., trypsin, plasmin, kallikrein, thrombin, etc.). Koy, C, et al. (Sulfur Letters 21:75- 88, 1998) discuss the synthesis of binol sulfate and its reactivity towards primary, secondary, and tertiary amines. Schafer & Tilley (Journal of the American Chemical Society 123:2683- 2684, 2001) discuss the efficient and high-yield synthesis of multigram quantities of various chiral BINOL (l,l'-Bi-2-naphthol )-containing macrocyles using zirconocene coupling, and observing that zirconocene coupling directed syntheses proceed in a highly diastereo selective fashion to yield homochiral products). Cabri, W., et al. (Tetrahedron Letters 32:1753-1756, 1991) discuss the use of a catalyst comprising Pd(OAc)₂ and l,3-bis(diphenylphosino)propane (DPPP) for arylation (e.g., with 1-naphthyl derivatives: aryl halides; aroyl chlorides; or aryltrifluormethane sulfonates) of vinyl butyl ether with high α-selectivity. Zewge, D., et al (Tetrahedron Letters 45:3729-3732, 2004) discuss the efficient use of SiCL/Lil in the presence of catalytic amounts of BF3 to debenzylate various compounds, including debenzylation of 6- methoxy-2-naphthonitirile. Parac-Vogt, & Binnemans (Tetrahedron Letters 45:3137-3139, 2004) discuss the use of lanthanide(III) nosylates (prepared from p-nitrotoluenesulfonic acid and the corresponding lanthanide(III) oxide) as recyclable catalysts for nitration of simple aromatic compounds, including nitration of naphthalene. Cui, Y., et al (Inorganic Chemistry 41:1033- 1035, 2002) discuss the synthesis of a rigid angular bridging ligand (7-oxa-dibenzofluorene- 3,11-dicarboxylic acid (H₂L)) by cyanation of rαc-6,6'-dibromo-l,l'-bi-2-naphthol, followed by ring closure and hydrolysis with sulfuric acid, and further discuss the self-assembly of such ligands to form nanoscopic molecular rectangles and coordination polymers containing open channels. Van Veldhuizen, JJ. , et al (Journal of the American Chemical Society 125:12502- 12508, 2003) discuss synthesis and characterization of six enantiomerically pure Ru-based metathesis catalysts (Ru carbenes that are chiral catalysts), based on modification of the benzylidine and chiral ligands of a prior styrenyl ether carbine that comprises a 1, l'-bi-naphthyl ring system. All of the preceding references are incorporated by reference herein in their entireties.

Scheme 2. Synthesis of regioisomers to 6,6'-dibromo-l,l'-bi-2-naphthol ((+)-25)

A) Synthesis of 3,3'-dibromo-l,l'-bi-2-naphthol (29)

B) Synthesis of 4,4'-dibromo-l,l'-bi-2-naphthol (31)

C) Synthesis of 5,5'-dibromo-l,l'-bi-2-naphthol (34)

D) Synthesis of 7,7'-dibromo-l,l'-bi-2-naphthol (37)

E) Synthesis of 8,8'-dibromo-l,l'-bi-2-naphthol (37)

Scheme 3. Synthesis of tetrabromo derivatives of l,l'-bi-2-naphthol (1) 2

3

4

O Scheme 4. Synthesis of 6,6'-difluoro and 6,6'-diiodo derivatives of l,l'-bi-2-naphthol (11)

A) Synthesis of 6,6'-difluoro-l,l'-bi-2-naphthol (48)

B) Synthesis of 6,6'-diiodo-l,l'-bi-2-naphthol (49)

O

Table 1. Exemplary species of the inventive compound genera.

Salts. The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge, S.M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

Preferably, the inventive compounds are non-charged, or neutral. Where particular compounds are charged, the neutral forms of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

EXAMPLE 1

(A HST2-based assay was developed to identify SIR2 inhibitors) Deacetylase protein. HST-I, -2, -3 and -4 are the four SIR2 homologs existing in yeast.

A plasmid containing the His-tagged catalytic domain of HST2 was obtained from Rolf Sternglanz (State University of New York, Stony Brook). His-tagged HST2 was over-expressed using BL21(DE3), and purified by Ni-NTA affinity chromatography (Qiagen, Valencia, CA). Highly purified HST2 was obtained using applicants' protocol developed for the purification of bacterially expressed His-tagged CtBP (Fjeld et al ). Briefly, the protein was applied to a Ni- NTA column, and the column was washed with successive gradients of glycerol (15-40%, 250 ml) and sodium chloride (50-350 mM, 250 ml). Protein was eluted from the column using an imidazole gradient (0-250 mM, 200 ml).

A charcoal-binding assay, also developed by applicants (Jackson 2003), was used to evaluate compounds for HST2 inhibitory activity. This assay is rapid, sensitive, and monitors the formation of O-Acetyl-ADPribose by measuring the amount of free [2-³H₃]acetate released upon hydrolysis of the enzymatic product. Because esters are susceptible to hydrolysis at high pH, free acetate is liberated only from O-Acetyl-ADPribose and not the acetylated lysine substrate. The [³H] -labeled acetylated peptide is removed from the reaction using activated charcoal, and the supernatant is counted using a liquid scintillation counter to determine the amount of free acetate. A 20-mer peptide corresponding to the terminal 20 amino acids of histone H3 (NH2-ARTKQTARKSTGGKAPRKQL-COOH) was obtained from SynPep (Dublin, CA), and the tritiated peptide substrate was generated using a modified protocol from Upstate Biotechnology (Lake Placid, NY). Particular modifications of the acetylation reaction were made to overcome difficulties encountered with peptide insolubility using the standard procedure. Specifically, the peptide was dissolved in DMSO, the amount of coupling reagent (BOP) was increased to increase the efficiency of the reaction and obtain labeled peptide with higher specific activity. Reaction products were purified by HPLC, with the acetylated peptides eluting at 25 to 30 minutes. Typically, approximately 75% of the amines within the peptide are labeled with [2-3[³H]]acetate, based upon the amount of radioactivity recovered in the purified peptide.

Data is plotted as percent remaining activity, comparing a DMSO control to reactions containing fixed concentrations of inhibitor.

EXAMPLE 2

(The importance of bi- and tri-cyclic ring systems, and in particular of the naphthyl ring moiety, in effective small molecule inhibitors of SIR2 was demonstrated)

Two initial approaches were used to identify novel SIR2 inhibitors. First, transition state analogs were designed and tested based on applicants' perception and understanding of the catalytic mechanism of SIR2. Second, applicants identified novel inhibitors based on perception and exploitation of similarities in the catalytic mechanism of SIR2 and PARP proteins. This second approach was further facilitated by the availability of several PARP inhibitors.

Inhibitor identification based on SIR2 transition- state analogs: First, and according to the present invention, novel SIR2 inhibitors were identified by designing and synthesizing transition state analogues, based on applicants' current understanding of the catalytic mechanism.

Specifically, Cholera Toxin subunit A was used to ADP ribosylate agmatine (decarboxylated arginine). For HST2, the IC50 of ADP ribosylated agmatine was determined to be -150 μM.

Inhibition studies showed that ADP ribosylated agmatine inhibited competitively with respect to peptide substrate, indicating that the inhibitor binds to the peptide-binding site. Additionally, several arginine derivatives were tested directly as inhibitors of SIR2 at a concentration of 2 mM using the charcoal-binding assay (Figure 3) to determine which arginine derivative may be a better SIR2 inhibitor when ADP ribosylated.

Surprisingly, L-arginine b-naphthylamide was found to be significantly better than the other arginine compounds tested, indicating, according to the present invention, the importance of the naphthyl ring for SIR2 binding. Interestingly, L-arginine β-naphthylamide was found to inhibit noncompetitively with respect to peptide, indicating that L-arginine β-naphthylamide binds to a site that is distinct from that bound by acetylated peptide and ADP ribosylated agmatine. However, without being bound by mechanism, ADP ribosylating L-arginine β- naphthylamide may result in a SIR2 inhibitor that binds to two separate sites, whereas a single- site inhibitor would be preferred.

Inhibitor identification based on perception of shared catalytic mechanism between SIR2 and PARP proteins: Second, and according to the present invention, novel SIR2 inhibitors were identified, by exploiting applicants' appreciation of the similar catalytic mechanism shared by SIR2 proteins and PARP proteins (Figure 4); a process facilitated by the availability of several PARP inhibitors. Both SIR2 and PARP protein families are NAD⁺ hydrolases and release free nicotinamide. SIR2 proteins catalyze the attack of acetylated lysine on the Cl position of ADPribose, whereas PARP proteins catalyze the attack of protein glutamate on the Cl position of ADPribose. With SIR2, acetate is transferred from acetylated protein to ADPribose, whereas the PARPs transfer the ADPribose to the glutamate containing substrate protein. This mechanism-based approach was pursued to test candidate compounds for SIR2 inhibition activity, and identify chemical components important for binding to SIR2. The least effective inhibitors were nicotinamide -based, benzamide and 3- aminobenzamide. Figure 5 shows that at 200 μM, the PARP inhibitors APlOO and APlOl were significantly more effective than the PARP inhibitors PJ34 and DPQ. Comparison of these compounds indicated that the aliphatic portions present in PJ34 and DPQ are deleterious for SIR2 binding and indicating the importance of the simple bi and tricyclic ring systems for SIR2 binding. Significantly, according to the present invention, the presence of a naphthyl moiety in the most effective PARP inhibitor (APlOl) was recognized.

EXAMPLE 3 (Compounds containing two naphthyl ring systems (binaphthyl compounds) were found to be significantly better at inhibiting SIR2 than compounds containing only one such system)

The results disclosed above under EXAMPLE 2, relating to two separate approaches taken by applicants (i.e., SIR2 transition state analogs, and compounds implicated based on shared catalytic mechanism between SIR2 and PARP), show according to the present invention that the naphthyl ring system is substantially important for SIR2 binding.

Therefore, a series of naphthyl containing compounds (at 286 μM), including disubstituted amino naphthols and binaphthol, was thus further investigated (Figure 6) to identify important determinants for SIR2 binding. Significantly, the compound containing two naphthol rings ((l,l'-binaphthyl)-2,2'-diol; compound 1) was significantly better at inhibiting HST2 than the amino naphthol compounds.

EXAMPLE 4

(SIR2 binding/inhibition activity was found to be sensitive to both the aromatic nature and the conformation of the binaphthyl ring system, and additionally was found to be sensitive to the identity of substituents at the 2, 2' positions)

Applicants' results of EXAMPLE 3 indicate that compounds containing two naphthol rings will be significantly better at inhibiting SIR2 than analogous single ring compounds. Therefore, a series of binaphthyl compounds was tested to further investigate important determinants for binding.

FIGURE 7 shows a comparison, according to the present invention, of SIR2 inhibition activity among a series of binaphthyl compounds (at 143 μM). SIR2 binding/inhibition activity was found to be sensitive to both the aromatic nature and the conformation of the binaphthyl ring system, and additionally was found to be sensitive to the identity of substituents at the 2, 2' positions. For example, the loss of one aromatic ring in each naphthyl moiety (as represented by the "S(-) Oct" and the biquinone ("biqui") compounds) more than doubled the HST2 activity (resulted in a poorer inhibitor). Additionally, substituting the alcohols of binol with amines

5 (e.g., as in (l,l'=binaphthyl)-2,2'-diamine)) yielded approximately a four-fold increase in HST2 activity (resulted in a poorer inhibitor) (e.g., compare compounds "R(+) OH" and "S(-) OH" with the "R(+) NH" and "S(-) NH"compounds). Moreover, addition of phosphate at the 2,2' position (e.g., as in 4-Hydroxydinaphtho(2,l-d: l',2'-f)(l,3,2)dioxaphosphepin 4-oxide (aka 1,1'- Binaphthyl-2,2'-diyl hydrogen phosphate)) prevents rotation at the intranaphthyl bond, and

O resulted in a 3-4 fold loss in HST2 activity (resulted in a better inhibitor) compared to binol and indicates that SIR2 inhibition activity favors a planar conformation.

The substituents at the 2, 2' position thus influence compound binding to SIR2. Without being bound by mechnanism, there may be important hydrogen bonding interactions with SIR2 binding site residues or, the substituents influence on the conformation of the binaphthyl rings

5 may be more important. Taken together, the structure/activity relationships of FIGURE 7 suggest that the influence of the substituents on the electron density of the naphthyl rings is a major factor in binding. The increased inhibition observed with hydroxyl versus amine functional groups indicates that SIR2 prefers binaphthyl compounds that are more electron deficient. Consistent with this, the binol derivative containing the strong electronegative Br

O groups was found to be the most potent SIR2 inhibitor analyzed (FIGURE 5).

EXAMPLE 5

(The influence of substituents on electron density of the naphthyl rings was found to be a major factor in SIR2 binding, and indicates that SIR2 prefers binaphthyl compounds that are more electron deficient)

Applicants' results of EXAMPLE 4 indicated that the substituents at the 2, 2' position influence compound binding to SIR2. Without being bound by mechnanism, there may be important hydrogen bonding interactions with SIR2 binding site residues or, the substituents influence on the conformation of the binaphthyl rings may be more important. Taken together, the structure/activity relationships of EXAMPLE 4 and FIGURE 7 indicated that the influence of the substituents on the electron density of the naphthyl rings is a major factor in binding. The increased inhibition observed with alcohol over amine indicates that SIR2 prefers binaphthyl compounds that are more electron deficient. Consistent with this premise, Figure 8 shows that a binol derivative (dibromo naphthol; tested at 50 μM) containing strong electronegative bromine (Br) groups was found to be a particularly potent SIR2 inhibitor. Likewise, according to the present invention, cyano groups and halogenated groups are also useful in providing potent SIR2 inhibitors. The influence of substituents on electron density of the naphthyl rings was thus found to be a major factor in SIR2 binding, and indicates that SIR2 prefers binaphthyl compounds that are more electron deficient.

EXAMPLE 6

(Inventive novel SIR2 inhibitors CBN and DBN were shown to substantially increase p53 acetylation in intact cells, thus supporting therapeutic utility)

The above EXAMPLES 1-5 show, according to the present invention, that binaphthyl- based compounds are preferred strong SIR2 inhibitors. According to the present invention, therefore, such inhibitors have substantial utility to inhibit SIR2-mediated deacetylation of p53. Experiments were therefore performed to confirm that the inventive SIR 2 inhibitors could substantially increase p53 acetylation in intact cells, thus supporting therapeutic utility.

Figures 9A and 9B show the results of a representative H1299 cell-based assay for K382 (lysine 382) acetylation of p53. H1299 cells were extracted 1 hr after treatment with 5 mM nicotinamide ("N"; known to inhibit both Sir2 and SIRTl in vitro; Bitterman et al., J Biol Chem. 22;277:45099-107, 2002), 1 μM TSA ("T"; Trichostatin A, an HDAC inhibitor), varied concentrations (0.2, 2, 20 or 200 μM) of either CBN (6,6'-Dicyano-l,l'-bi-2-naphthol; compound 5) or DBN ((S)-6,6'-Dibromo-l,l'-bi-2-naphthol; compound 2) in the presence of TSA ("T+CBN" and "T+DBN"), and the combination (as a positive control) of 5 mM nicotinamide and 1 μM TSA ("T+N"). Acetylated levels of p53 on K382 were detected using the antibody from cell signaling ("p53 K382"). Total p53 levels were measured using DOl antibody ("p53 Total").

FFIGURE 9B shows that CBN and DBN increase p53 acetylation in intact H1299 cells. FIGURE 9A shows that the IC₅₀ values were < 1 μM.

EXAMPLE 7

(The inventive l,l'-bi-naphthalene-based inhibitors in were shown to be effective in promoting BCL6 acetylation in an art-recognized model of NHL) This Example discloses experiments conducted to demonstrate that the inventive 1,1 '-bi- naphthyl-based inhibitors promote BCL6 acetylation in a model of non-Hodgkin's lymphoma (NHL).

The proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is necessary for germinal-center formation and has been implicated in the pathogenesis of B-cell lymphomas. The co-activator p300 binds and acetylates BCL6 in vivo and inhibits its function. Acetylation disrupts the ability of BCL6 to recruit histone deacetylases (HDACs), thereby hindering its capacity to repress transcription and to induce cell transformation. BCL6 is acetylated under physiologic conditions in normal germinal-center B cells and in germinal center- derived B-cell tumors. Treatment with specific inhibitors shows that levels of acetylation of BCL6 are controlled by both HDAC-dependent and SIR2-dependent pathways. Pharmacological inhibition of these pathways leads to the accumulation of the inactive acetylated BCL6 and to cell-cycle arrest and apoptosis in B-cell lymphoma cells (Bereshchenko et al (Nat Genet., 32:606-13, 2002).

Ramos cell model of NHL. Burkitt's lymphoma is one type of a group of malignant diseases know as the Non-Hodgkin's Lymphomas (NHL). Ramos (RA 1) cells are an in vitro line derived from an American Burkitt lymphoma, designated Ra No. 1, which produced malignant tumors when inoculated into thymus-deficient nude mice (ATCC No. CRL- 1596; see, e.g., Benjamin D, et al. J. Immunol. 129:1336-1342, 1982; Klein G, et al. Intervirology 5:319- 334, 1975; Rousset F, et al. J. Immunol. 140:2625-2632, 1988; Nilsson K, et al. Int. J. Cancer 19:337-344, 1977; Bloom TJ, Beavo JA Proc. Natl. Acad. ScL USA 93:14188-14192, 1996). The cells have B-lymphocyte characteristics, with surface- associated mu and kappa chains and Epstein-Barr virus (EBV) receptors, and can be readily infected with EBV in vitro.

Methods. The acetylation state of endogenously expressed BCL6 in Ramos cells was investigated by the current applicants to determine the ability of the inventive 1,1' -bi- naphthalene-based inhibitors to inhibit the NAD+-dependent deacetylase activity of SIRTl in a model of non-Hodgkin's lymphoma (NHL). Ramos cells were treated with DBN (6,6'-

Dibromo-l,l'-bi-2-naphthol; compound 2) alone or in combination with trichostatin A ("TSA," an HDAC inhibitor)). For comparison, Ramos cells were also treated with TSA, nicotinamide, and, as a positive control, TSA in combination with nicotinamide. The acetylation state of BCL6 was determined by immunoprecipitation followed by western blotting (FIGURE 10) using an antibody against acetyl-lysine as described by Bereshchenko et al (Nat Genet., 32:606- 13, 2002).

As shown in FIGURE 10, BCL6 acetylation was elevated in cells treated with the combination of TSA (lμM) and nicotinamide (5 mM) as compared to TSA or nicotinamide alone. Notably, treatment with TSA and nicotinamide individually had relatively little effect on BCL6 acetylation. The acetylation state of BCL6 in cells treated with DBN (500 nM) alone was comparable to that seen in cells treated with the combination of TSA and nicotinamide. By far the most robust elevation in BCL6 acetylation was observed in cells treated with DBN in combination with TSA. These observations demonstrate further utility of the inventive binaphthyl-containing inhibitors in vivo (NHL model system) and indicate that DBN is not only cell-permeable, but likely has an even lower IC₅₀ value than that observed in in vitro assays.

EXAMPLE 8

(Substantial utility in promoting apoptosis)

According to the present invention, the inventive binaphthyl-containing inhibitors have substantial utility in promoting apoptosis. As discussed in the above Examples and tissue culture models, the acetylation of BCL6 and p53 could be increased with DBN (e.g., in the range of about 150 to about 200 nM). Additionally, and most importantly, apoptosis (cell death) of non-Hodgkin's lymphoma cells was promoted by DBN administration, but not in control fibroblasts at similar doses. Applicants have further determined that the efficacy of DBN in promoting maximal protein acetylation or cell death (apoptosis) can be enhanced by administration of etoposide (an inhibitor of topoisomerase 2) and/or trichostatin (an inhibitor of Type I and Type II histone deacetylases, or HDACs). Alternately, according to particular aspects of the present invention, DBN markedly increases the effects of topoisomerase 2 inhibitors and/or Type I and Type II HDACs on non- Hodgkin's lymphoma cells.

Animal model studies. An exemplary experimental model involves the intravenous injection of SCID mice with Ramos cells (the same type of non-Hodgkin's lymphoma cells used in tissue culture studies elsewhere herein. Mice injected with these cells succumb to hind limb paralysis within 20 days due to massive spread of lymphoma cells within the retroperitoneal space. Among a variety of administration paradigms, doses of DBN approaching those found to be active on tissue culture could be obtained by delivering the agent in subcutaneous silastic tubes. DBN levels in plasma were monitored using HPLC and mass spectroscopy. Insertion of two silastic tubes, 3 cm in length, could deliver doses of DBN of greater than 200 nM in plasma for periods of at least 4 weeks.

Because HDAC inhibitors suitable for treating animals are not available commercially, so SAHA, a Type I and II HDAC inhibitor, was synthesized for administration in conjuction with DBN. . SAHA is relatively non-toxic, and has recently been used clinically for treatment of other malignancies. SAHA, dissolved and diluted in DMSO, was synthesized as previously described (e.g., Richon et al, Proc. Natl. Acad. ScL USA 93:5705-5708, 1996).

Surprisingly, in animal model studies, SAHA markedly increased the bioavailability of DBN by over two-fold. Mice given DBN alone achieve levels of 380 nM, while mice given DBN and SAHA had DBN levels of 840 nM. Without being bound by mechanism, SAHA presumably inhibits an enzyme that degrades DBN. Regardless of mechanism, SAHA has substantial utility for helping to achieve and/or maintain efficacious DBN levels.

Therefore, particular aspects provide methods of treatment (e.g.,: cell proliferative disorders conditions (e.g., tumors); activities mediated by cellular NAD+-dependent deacetylase activity; HIV infection or a HIV-related cellular effects, conditions or diseases), comprising administration of a therapeutically effective amount of a compound of the present invention, and further comprising administration of an inhibitor of topoisomerase 2 (e.g., etoposide), an inhibitor of type I and/or II histone deacetylase (HDAC) (e.g., suberoylanilide hydroxamic acid (SAHA)), or both.

EXAMPLE 9

(Substantial utility for inhibiting HIV transcription; efficacy in model of HIV)

According to the present invention, the inventive binaphthyl-containing inhibitors (1,1'- binaphthyl-based compounds) have substantial utility in inhibiting HIV transcription, and for treatment of HIV-related cellular effects, conditions and diseases conditions, including AIDS, and the inventive (l,l'-binaphthyl-based compounds) have substantial utility for treatment or prevention of HIV infection and/or replication and HIV-related cellular effects, conditions and diseases. The phrase "HIV-related cellular effects, conditions and diseases" or "HIV-related (or mediated) conditions or diseases" refers to those illnesses and conditions included in, but not necessarily limited to the CDC 1993 AIDS surveillance case definition, as follows: Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer, invasive *; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5 C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HIV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1 month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HIV.

EXAMPLE 10

(Malaria therapeutic agents; substantial utility of Sir2 inhibitors for inhibiting malaria; utility as immunotherapeutic agents against malaria.)

Novel therapeutic agents to treat Malaria. The protein deacetylase Sir2 plays an important role in protecting rapidly dividing cells from the deleterious affects of DNA damage that incur during proliferation. It is not well understood how Sir2 promotes survival of these cells although many studies demonstrate that the enzymatic activity of Sir2, important for DNA packaging, is critical for viability. This feature of Sir2 makes it a very promising drug target for major life-threatening diseases involving rapidly dividing cells such as cancer and infectious diseases caused by parasites and bacteria.

In the case of malaria, two Australian research groups have reported that Plasmodium falciparum Sir2 (PfSir2) has a specialized role in keeping the parasite invisible to the host by regulating the expression of the variable, or var, genes. The var genes encode proteins known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-I). According to particular aspects of the present invention, the inventive binaphthyl- containing inhibitors (l,l'-binaphthyl-based compounds) have substantial utility for treatment of malaria; for example, as immunotherapeutic agents against malaria.

In certain embodiments, DBN inhibits PfSir2 and prevents the mono-allelic expression of the var genes. In certain aspects, DBN induces the expression of multiple var genes. For example, according to certain aspects, treating malaria-infected red blood cells with DBN and measuring gene expression by Northern blot shows expression of multiple var genes. In other aspects, involving genetic approaches to show that the enzymatic activity of PfSir2 is required for controlling var gene expression, a catalytically inactive PfSir2 protein is overexpressed in stably transfected parasites and gene expression is measured by Northern blot, whereby the detection of multiple var genes in response to DBN treatment indicates that the inventive Sir2 inhibitors expose malaria to the host immune system to allow for removal of infected red blood cells by splenic clearance.

EXPERIMENTAL:

Rationale. The Cowman Lab demonstrated that disrupting the PfS IR2 gene results in the expression of multiple var genes. This was observed by microarray analysis and Northern blot {Cell. 121:13-24, 2005). The present applicants therefore tested to see if inhibiting the enzymatic activity of PfSIR2 has the same effect on expression of multiple var genes as knocking out the PfSIR2 gene.

SIR2 Inhibitors as immunotherapeutic agents against malaria. A genetic approach to address this question was taken to stably transfect the parasite with a dominant negative PfSIR2. A dominant negative PfSIR2 was generated by mutating the catalytic Histidine to Tyrosine. GFP was then replaced by mutant PfSIR2 H132Y in a suitable expression vector (FIGURE 11). Applicants mutated the catalytic Histidine to a Tyrosine and inserted the gene into a vector obtained from Brendan Crabb's lab. (contains a Toxoplasma gondii transactivator) (Proc Natl Acad Sci U S A.; 102:2980-5, 2005).

Figure 11 shows, according to particular aspects of the present invention, a dominant negative PfSIR2 generated by mutating catalytic Histidine to Tyrosine. GFP was replace by the mutant (PfSIR2 H 132Y) in an expression vector obtained from the Crabtree lab.

Asexual, blood stage parasites were transfected with the vector and cultured in red blood cells under constant drug pressure (ATC and WR99) (R99210 selects for transfected parasites). The experiment was initiated by splitting the culture, removing ATC from one, and culturing a nontransfected control alongside. ATC (anhydrotetracycline) is included to prevent expression. In this system, removal of ATC allows for expression of PfSIR2 H132Y.

RESULTS:

A Northern Blot revealed the induction of multiple var genes in both transfected cultures suggesting a lack of tetracycline control. Selective PCR using a primer specific to the mutant gene confirms the presence of the dominant negative PfSIR2 transcript in both the un-induced and induced cultures (compare "C" with "U" and "I" in Figure 12). Figure 12 shows Northern blots probed with Exon 2 probe, which Hybridizes to conserved region present in many var genes (C=control; U=un-induced (+ATC); and I induced (-ATC). Figure 13 shows, according to exemplary aspects of the present invention, selective PCR confirmation of expression of dominant negative PfSIR2 in transfected cultures.

Significantly, this dominant negative experiment demonstrates that a small molecule inhibitor of PfSIR2 can reasonable serve to expose the parasite to the host by inducing the expression of multiple var genes. Applicants determined that DBN kills the parasite at 5uM in about 3 days and that the parasites don't grow very well at IuM DBN.

EXAMPLE 11

(Substantial utility of Sir2 inhibitors as agents against infectious diseases: Trypanosoma brucei (African sleeping sickness; Leishmaniasis (e.g., Leishmania infantum, etc.); Mycobacterium tuberculosis; and Anthrax)

The protein deacetylase Sir2 plays an important role in protecting rapidly dividing cells from the deleterious affects of DNA damage that incur during proliferation. It is not well understood how Sir2 promotes survival of these cells although many studies demonstrate that the enzymatic activity of Sir2, important for DNA packaging, is critical for viability. This feature of Sir2 makes it a very promising drug target for major life-threatening diseases involving rapidly dividing cells such as cancer and infectious diseases caused by parasites and bacteria.

Trypanosoma brucei (African sleeping sickness):

According to particular aspects of the present invention, the inventive binaphthyl- containing inhibitors (l,l'-binaphthyl-based compounds) have substantial utility for treatment of African sleeping sickness (Trypanosoma brucei). Genetic studies in Trypanosoma brucei, the causative agent of African sleeping sickness, have indicated that Sir2 is critical for viability in the presence of a DNA-damaging agent. Therefore, in particular exemplary embodiments, DBN has substantial utility as an antitrypanosomal agent. For example, Applicants have used an initial a spectrophotometric assay to show that a Sir2 inhibitor kills Trypanosoma brucie in culture at inhibitor concentrations obtainable in mice -500 nM. Alternatively, more exact assays, for example cell counting are used to show this efficacy. In particular exemplary aspects, the Sir 2 inhibitor is DBN and/or other molecules disclosed herein. In certain exemplary aspects, the Sir2 ihibitors {e.g., DBN, and/or other molecules disclosed herein) are potent agent against Trypanosoma brucie at micromolar, and/or at namomolar concentrations. Preferably, the Sir2 ihibitors {e.g., DBN, and/or other molecules disclosed herein) are potent agent against Trypanosoma brucie at namomolar concentrations. In certain exemplary aspects, the Sir2 ihibitors {e.g., DBN, and/or other molecules disclosed herein) have efficacy in a mouse model of sleeping sickness.

Leishmaniasis (Leishmania infantum^:

The commercially available Sir2 inhibitor, SIRTINOL™ has been reported to inhibit in vitro proliferation of Leishmania infantum axenic amastigotes at 30 uM (). Applicants' studies with DBN have revealed that the exemplary inventive inhibitor DBN is about 5 -fold more potent than SIRTINOL™ in effecting such inhibition. According to particular embodiments, DBN and derivatives and/or anlalogs of DBN are effective at killing the parasite at nanomolar concentrations, and thus the inventive binaphthyl-containing inhibitors (l,l'-binaphthyl-based compounds) {e.g., DBN, and/or other molecules disclosed herein) have substantial utility for treatment of Leishmaniasis {Leishmania infantum).

Mycobacterium TuberculoύslMycobacterium tuberculosis):

According to additional inventive aspects, the inventive binaphthyl-containing inhibitors (l,l'-binaphthyl-based compounds) {e.g., DBN, and/or other molecules disclosed herein) have substantial utility for treatment of Mycobacterium, including the severe pathogen of Mycobacterium tuberculosis; that is, Sir2 inhibitors have utility as an anti-TB drugs. In particular embodiments the inventive Sir2 inhibitors have utility for treating the closely related, nonpathogenic fast-growing model organism, Mycobacterium smegmatis. In preferred embodiments, the inventive Sir2 inhibitors have utility for treating Mycobacterium tuberculosis.

According to particular aspects, a major obstacle for drug design approaches targeting Mycobacterium is the waxy wall that protects the organism; that is penetrating the bacterial coat Therefore, in particular aspects, derivatives of DBN that are more hydrophobic are preferred, and along with DBN, exemplary species of such more hydrophobic derivatives have been synthesized by Applicants:

For example, according to particular aspects, the sulfate and methylene derivatives are significantly more hydrophobic, and effective in penetrating the waxy coat than DBN, and yet retain the bromines at the 6,6' position which has proven to be critical for high affinity binding. According to particular aspects, the inventive Sir2 inhibitors, and particularly the more hydrophobic derivatives thereof, are effective in inhibiting Mycobacterium smegmatis growth, and are effective in inhibiting Mycobacterium tuberculosis.

Anthrax:

Applicants have, as described herein, identified a novel inhibitor for the NAD-dependent deacetylase SIR2 with nanomolar potency against yeast and human enzymes. Anthrax is a major problem, and while several antibiotics, including penicillin, are effective against most strains, there is concern about emergence of antibiotic-resistant strains. Moreover, spores are not targets of inhibitors as they are dormant and not carrying out enzymatic reactions. There is, therefore, a pronounced need in the art for novel methods and agents for inhibiting Anthrax. The Bacillus anthracis SIR2 protein has recently been cloned (). According to additional inventive aspects, just as in the case of organisms such as leishmania and trypanosome, SIR2 is critical for the viability of Anthrax (e.g., Bacillus anthracis), and the inventive binaphthyl- containing inhibitors (l,l'-binaphthyl-based compounds) (e.g., DBN, and/or other molecules disclosed herein) have, therefore, substantial utility for treatment of Anthrax.

As appreciated in the art, various concentrations of the drug are tested for efficacy in growth inhibition assays (e.g., on plates and/or in broth, etc) using representative strains to determine an effective inhibitor concentration for preventing growth (e.g., on agar plates, etc.), or to determine an effective inhibitor concentration for slowing growth in solution cultures, or to determine an effective inhibitor concentration for killing, for example, Bacillus anthracis.

According to particular aspects, an effect in slowing growth or killing in the low micromolar range confirms that SIR2 is the drug target. According to additional aspects, recombinant SIR2 protein has utility to confirm efficacy of the inventive derivative compounds and/or to confirm agents having enhanced potency relative to others. Alternatively, efficacy utility is confirmed by screening the derivatives directly against the organism.

According to further aspects, an effect in slowing growth or killing in the low to mid nanomolar range confirms the potential of the inventive compound as a therapeutic agent.

Use of the inventive SIR2 inhibitors in combination with an agent that causes DNA damage: In cancer models, SIR2 inhibitors have been coupled with topoisomerase inhibitors like etoposide. As discussed above, in many systems, SIR2 functions to prevent death in the midst of DNA damage. For example, in a trypanosoma study, a dominant negative SIR2 was unable to rescue death caused by a DNA-alkylating agent, whereas overexpression of WT SIR2 was able to rescue. In leishmania, a poor SIR2 inhibitor was effective alone. Therefore, according to additional aspects of the present invention, the inventive SIR2 inhibitors are used in combination with an agent that causes DNA damage.

Claims

1. A compound of Formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵, R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO, -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁶ is independently hydrogen, halogen, -NO₂, -CHO, -CFl₃, -CH₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein the compound is of Formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

3. The compound of claim 1, wherein the compound is of formula III:

wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl; wherein X is >C0, >S0₂ or >PO₂R⁹, wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl; and wherein Y and Z are O, except where X is CO wherein exactly one of Y or Z is methylene; and the pharmaceutically acceptable salts thereof.

4. The compound of claim 2, wherein the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as in claim 2.

5. The compound of claim 3, wherein the compound is of formula V:

wherein R _>3 , _π R4 , X, Y and Z are as defined in claim 3.

6. The compound of claim 2, wherein R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl.

7. The compound of claim 2, wherein the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'- Bis(methyl)-l,l'-bi-2-naphthol, 6,6'- Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2- naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'-Bis(trifluoromethyl)-l,l'- bi-2-naphthol sulfate.

8. A pharmaceutical composition, comprising a pharmaceutically acceptable diluent, carrier or excipient, along with a compound of Formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵, R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R is independently hydrogen, halogen, -NO₂, -CHO, -CFl₃, -CH₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

9. The pharmaceutical composition of claim 8, wherein the compound is of Formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci_₆alkyl, and the pharmaceutically acceptable salts thereof.

10. The pharmaceutical composition of claim 8, wherein the compound is of formula III:

11. The pharmaceutical composition of claim 9, wherein the compound is of formula IV:

wherein R¹, R², R³ and R⁴ are as in claim 9.

12. The pharmaceutical composition of claim 10, wherein the compound is of formula

V:

wherein R _>3 , _π R4 , X, Y and Z are as defined in claim 10.

13. The pharmaceutical composition of claim 8, wherein R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, - C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl.

14. The pharmaceutical composition of claim 8, wherein the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'- Bis(methyl)-l,l'-bi-2- naphthol, 6,6'-Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'- Dicyano-l,l'-bi-2-naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol sulfate.

15. The pharmaceutical composition of claim 8, further comprising an amount of an antineoplastic agent.

16. The pharmaceutical composition of claim 15, wherein the antineoplastic agent is selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites, antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological response modifiers, DNA- alkylating agents, DNA intercalators, DNA repair agents, growth factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disrupters and topoisomerase I and II inhibitors, and combinations thereof.

17. The pharmaceutical composition of claim 15, wherein the antineoplastic agent is selected from the group consisting of cyclophosphamide, triethylenephosphoramide, triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine, trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU, benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil, cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin, estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide, improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine, vincristine, vinorelbine and vindesine.

18. A method for treating a cell proliferative disorder, comprising administering, in a subject in need thereof, a therapeutically effective amount of a compound of Formula I:

19. The method of claim 18, wherein the compound is of Formula II:

20. The method of claim 18, wherein the compound is of Formula III:

21. The method of claim 19, wherein the compound is of Formula IV:

wherein R¹, R², R³ and R are as in claim 19.

22. The method of claim 20, wherein the compound is of Formula V:

wherein R _>3 , _π R4 , X, Y and Z are as defined in claim 20.

23. The method of claim 18, wherein R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl.

24. The method of claim 18, wherein the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'- Bis(methyl)-l,l'-bi-2-naphthol, 6,6'- Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2- naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, 6,6'-Bis(trifluoromethyl)-l,l'-bi- 2-naphthol sulfate, and combinations thereof.

25. The method of claim 18, wherein the cell proliferative disorder is cancer.

26. The method of claim 18, wherein the cancer is selected from the group consisting of Non-Hodgkins lymphoma, B-cell-derived Non-Hodgkins lymphoma, diffuse large B-cell lymphoma, CML, and combinations thereof.

27. The method of claim 18, further comprising administration of an antineoplastic agent.

28. The method of claim 27, wherein the antineoplastic agent is selected from the group consisting of antiangiogenic and antivascular agents, antimetabolites, antifolates and other inhibitors of DNA synthesis, antisense oligonucleotides, biological response modifiers, DNA- alkylating agents, DNA intercalators, DNA repair agents, growth factor receptor kinase inhibitors, hormone agents, immunoconjugates, microtubule disrupters and topoisomerase I and II inhibitors, and combinations thereof.

29. The method of claim 27, wherein the antineoplastic agent is selected from the group consisting of cyclophosphamide, triethylenephosphoramide, triethylenethiophosphoramide, flutamide, altretamine, triethylenemelamine, trimethylolmelamine, meturedepa, uredepa, aminoglutethimide, L-asparaginase, BCNU, benzodepa, bleomycin, busulfan, camptothecin, capecitabine, carboquone, chlorambucil, cytarabine, dactinomycin, daunomycin, daunorubicin, docetaxol, doxorubicin, epirubicin, estramustine, dacarbazine, etoposide, fluorouracil, gemcitabine, hydroxyurea, ifosfamide, improsulfan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, novembrichin, paclitaxel, piposulfan, plicamycin, prednimustine, procarbazine, tamoxifen, temozolomide, teniposide, thioguanine, thiotepa, UFT, uracil mustard, vinblastine, vincristine, vinorelbine and vindesine.

30. A method for modulating a condition or activity mediated by cellular N AD+- dependent deacetylase activity, comprising contacting a cell having NAD+-dependent deacetylase activity with a NAD+-dependent deacetylase inhibiting amount of a compound of formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵ R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, - CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁶ is independently hydrogen, halogen, -NO₂, -CHO, -CFl₃, -CH₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹ ,and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

31. The method of claim 30, wherein the compound is of Formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof; and whereby modulating of a condition or activity mediated by cellular NAD+-dependent deacetylase activity is afforded.

32. The method of claim 30, wherein the compound is of formula III:

33. The method of claim 31, wherein the compound is of formula IV: &

IV

6

wherein R¹, R², R³ and R⁴ are as in claim 31.

34. The method of claim 32, wherein the compound is of formula V:

wherein R _>3 , _π R4 , X, Y and Z are as defined in claim 32.

35. The method of claim 30, wherein R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci-βalkyl.

36. The method of claim 30, wherein the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'- Bis(methyl)-l,l'-bi-2-naphthol, 6,6'- Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2- naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, and 6,6'-Bis(trifluoromethyl)-l,l'- bi-2-naphthol sulfate.

37. The method of clam 30, wherein inhibiting the NAD+-dependent deacetylase activity comprises inhibiting activity of a member of the SIR2 family of protein deacetylases.

38. The method of claim 37, wherein modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises activating a silenced cellular gene.

39. The method of claim 37, wherein modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises promoting p53-dependent apoptosis.

40. The method of claim 37, wherein modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting BCL6 transcriptional repressor activity.

41. The method of claim 37, wherein modulating the condition or activity mediated by cellular NAD+-dependent deacetylase activity comprises inhibiting HIV TAT-mediated transcriptional activation activity.

42. A method for treating for treating or preventing HIV infection or a HIV-related cellular effect, condition or disease, comprising administering, in a subject in need thereof, an effective amount of a compound of formula I:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³, R⁴, R⁵ R⁷ and R⁸ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, - CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, wherein R⁶ is independently hydrogen, halogen, -NO₂, -CHO, -CFl₃, -CH₃, -CN, - C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted Ci-βalkyl, and the pharmaceutically acceptable salts thereof.

43. The method of claim 42, wherein the compound is of formula II:

wherein R¹ and R² are independently -OH, -SH, -NH₂, or are taken together, along with the carbon atoms to which they are bound and the position 1' and 1 carbons, to form a 7- membered heterocyclic ring that comprises up to three heteroatoms independently selected from O, S, N or P; and wherein R³ and R⁴ are independently hydrogen, halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, or -C(NH)NR⁹R⁹, and wherein R⁹ is independently H, -CH₃, or substituted or unsubstituted C_1-6alkyl, and the pharmaceutically acceptable salts thereof.

44. The method of claim 42, wherein the compound is of Formula III:

45. The method of claim 43, wherein the compound is of Formula IV:

wherein R¹, R², R³ an R⁴ are as in claim 43.

46. The method of claim 44, wherein the compound is of Formula V:

wherein R _>3 , _π R4 , X, Y and Z are as defined in claim 44.

47. The method of claim 42, wherein R¹ and R² are -OH, wherein R³ and R⁴ are independently halogen, -NO₂, -CHO; -CFl₃, -CH₃, -CN, -C(O)R⁹, C(O₂)R⁹, -C(NH)NH₂, and wherein R⁹ is H, -CH₃, or substituted or unsubstituted Ci_₆alkyl.

48. The method of claim 42, wherein the compound is selected from the group consisting of 6,6'-Dibromo-l,l'-bi-2-naphthol, 6,6'- Bis(methyl)-l,l'-bi-2-naphthol, 6,6'- Dibromo-l,l'-bi-2-naphthol sulfate , 6,6'-Dicyano-l,l'-bi-2-naphthol, 6,6'-Dicyano-l,l'-bi-2- naphthol sulfate , 6,6'- Bis(trifluoromethyl)-l,l'-bi-2-naphthol, 6,6'-Bis(trifluoromethyl)-l,l'-bi- 2-naphthol sulfate, and combinations thereof.

49. The method of claim 42, further comprising administration of an anti-HIV agent.

50. The method of claim 49, wherein the anti-HIV agent is selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non-nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (PIs); fusion inhibitors (FI), and combinations thereof.

51. The method of claim 50, wherein the nucleoside reverse transcriptase inhibitor (NRTI) is selected from the group consisting of: lamivudine and zidovudine; FTC, emtricitabine; lamivudine, 3TC; abacavir/ lamivudine; zalcitabine, ddC, dideoxycytidine; zidovudine, AZT, azidothymidine, ZDV; abacavir, zidovudine, and lamivudine; tenofovir disoproxil/emtricitabine; enteric coated didanosine; didanosine, ddl, dideoxyinosine; Didanosine (ddl) delayed release capsules; tenofovir disoproxil fumarate; stavudine, d4T; abacavir, and combinations thereof.

52. The method of claim 50, wherein the non-nucleoside reverse transcriptase inhibitor (NNRTI) is selected from the group consisting of: delavirdine, DLV; efavirenz; nevirapine, BI-RG-587, and combinations thereof.

53. The method of claim 50, wherein the protease inhibitor (PI) is selected from the group consisting of: Amprenavir; indinavir, IDV, MK-639; saquinavir mesylate, SQV; saquinavir; lopinavir and ritonavir; Fosamprenavir Calcium; ritonavir, ABT-538; atazanavir sulfate; nelfinavir mesylate, NFV, and combinations thereof.

54. The method of claim 50, wherein the fusion inhibitor (FI) comprises enfuvirtide, T-20.

55. The method of claim 42, wherein the HIV-related cellular effect, condition or disease is selected from the group consisting of: AIDS; Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer, invasive *; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5°C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HIV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1-month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HIV, and combinations thereof.

56. The method of any one of claims 18, 30 and 42, further comprising administration of an inhibitor of topoisomerase 2.

57. The method of claim 56, wherein the inhibitor of topoisomerase 2 is etoposide.

58. The method of any one of claims 18, 30 and 42, further comprising administration of an inhibitor of type I and/or II histone deacetylase (HDAC).

59. The method of claim 58, wherein the histone deacetylase (HDAC) inhibitor is suberoylanilide hydroxamic acid (SAHA).

60. A method for treating for treating or preventing Malaria, comprising administering, in a subject in need thereof, an effective amount of a compound of formula I of claim 18.

61. A method for treating for treating or preventing infectious disease, comprising administering, in a subject in need thereof, an effective amount of a compound of formula I of claim 18.

62. The method for treating for treating or preventing infectious disease of claim 61, wherein the infectious disease is at least one selected from the group consisting of: Trypanosoma bruceϊ (African sleeping sickness); Leishmaniasis (e.g., Leishmania infantum, etc.); Mycobacterium tuberculosis; and Anthrax.

63. The method of claim 62, wherein the infectious disease is Anthrax.